Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

Meloxicam ameliorates motor dysfunction and dopaminergic neurodegeneration by maintaining Akt-signaling in a mouse Parkinson's disease model.

A series of oxicam non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to be neuroprotective against 1-methyl-4-phenyl pyridinium in human neuroblastoma SH-SY5Y cells via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway independent of cyclooxygenase (COX) inhibition. The present study endeavored to establish this novel effect of meloxicam (MLX), an oxicam NSAID, in a mouse Parkinson's disease (PD) model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Male C57BL/6 mice, which received MPTP (30 mg/kg/day; s.c.) for 5 consecutive days (chronic model) with 10-day follow-up saline administrations, showed significant motor dysfunction in the pole test due to reduced tyrosine hydroxylase (TH) protein levels in the brain on day 16 after MPTP/saline treatment. Daily coadministrations of MLX (10mg/kg/day; i.p.) and MPTP for the first 5 days and follow-up 10 days with MLX administrations alone (MPTP/MLX treatment) significantly ameliorated MPTP-induced behavioral abnormalities in mice. Concomitant decreases of TH protein levels in the striatum and midbrain of MPTP/MLX-treated mice were not only significantly (p<0.01 and p<0.05, respectively) ameliorated but phosphorylated Akt (pAkt473) expression in the midbrain was also significantly (p<0.01) increased in the midbrain when compared with MPTP/saline-treated mice. These results suggest that MLX, an oxicam NSAID, attenuated dopaminergic neuronal death in the experimental MPTP-PD model by maintenance of the Akt-signaling. Oxicam NSAIDs may serve as potential drugs for PD treatment via a novel mechanism of action.

Estimation of the duration after methamphetamine injection using a pharmacokinetic model in suspects who caused fatal traffic accidents.

When the population parameters of drug pharmacokinetics in the human body system are known, the time-course of a certain drug in an individual can generally be estimated by pharmacokinetics. In the present two cases where methamphetamine abusers were suspected to have inflicted mortalities in traffic accidents, the time-elapse or duration immediately after methamphetamine injection to the time when the accidents occurred became points of contention. In each case, we estimated the time-course of blood methamphetamine after the self-administration in the suspects using a 2-compartment pharmacokinetic model with known pharmacokinetic parameters from the literatures. If the injected amount can be determined to a certain extent, it is easy to calculate the average time-elapse after injection by referring to reference values. However, there is considerable individual variability in the elimination rate based on genetic polymorphism and a considerably large error range in the estimated time-elapse results. To minimize estimation errors in such cases, we also analyzed genotype of CYP2D6, which influenced methamphetamine metabolism. Estimation based on two time-point blood samples would usefully benefit legal authorities in passing ruling sentences in cases involving similar personalities and circumstances as those involved in the present study.

HRD1 levels increased by zonisamide prevented cell death and caspase-3 activation caused by endoplasmic reticulum stress in SH-SY5Y cells.

Zonisamide, which is commonly prescribed at high doses (200-400 mg/day) for the treatment of partial seizures, has recently been used at a low dose (25 mg/day) for improving parkinsonian syndrome. However, the molecular mechanisms that underlie the antiparkinsonian effects of zonisamide have not been clarified. Here we show that low micromolar concentrations of zonisamide prevented cleavage of caspase-3 and cell death in human dopaminergic SH-SY5Y neuroblastoma cells that were subjected to endoplasmic reticulum stress induced by tunicamycin or 6-hydroxydopamine. Hypodense zonisamide increased the expression levels of SEL1L, which is known to stabilize the ubiquitin ligase HRD1. Indeed, upregulation of HRD1 protein was observed. Thus, the results of this study strongly suggest that low concentrations of zonisamide inhibit neuronal cell death by increasing HRD1 protein levels in patients with Parkinson's disease. Consequently, in addition to the treatment of Parkinson's disease, the therapeutic potential of zonisamide should be considered for the treatment of several neurodegenerative disorders with pathophysiological mechanisms involving endoplasmic reticulum stress.

[Longer working hours of pharmacists in the ward resulted in lower medication-related errors--survey of national university hospitals in Japan].

It is obvious that pharmacists play a critical role as risk managers in the healthcare system, especially in medication treatment. Hitherto, there is not a single multicenter-survey report describing the effectiveness of clinical pharmacists in preventing medical errors from occurring in the wards in Japan. Thus, we conducted a 1-month survey to elucidate the relationship between the number of errors and working hours of pharmacists in the ward, and verified whether the assignment of clinical pharmacists to the ward would prevent medical errors between October 1-31, 2009. Questionnaire items for the pharmacists at 42 national university hospitals and a medical institute included the total and the respective numbers of medication-related errors, beds and working hours of pharmacist in 2 internal medicine and 2 surgical departments in each hospital. Regardless of severity, errors were consecutively reported to the Medical Security and Safety Management Section in each hospital. The analysis of errors revealed that longer working hours of pharmacists in the ward resulted in less medication-related errors; this was especially significant in the internal medicine ward (where a variety of drugs were used) compared with the surgical ward. However, the nurse assignment mode (nurse/inpatients ratio: 1 : 7-10) did not influence the error frequency. The results of this survey strongly indicate that assignment of clinical pharmacists to the ward is critically essential in promoting medication safety and efficacy.

[Dissolution behaviors of tablet and capsule covered with oblate or agar jelly for taking medicine easily].

Drugs are sometimes covered with oblate or agar jelly. It is said that the medicinal effect of drugs covered with oblate is slow, but no studies have reported results confirming this. Therefore, we examined the dissolution behavior when the drug was covered with oblate or agar jelly. Three types of commercially available formulations of benzodiazepine were used: medazepam sugarcoated tablets, prazepam uncoated tablets, and clorazepate dipotassium capsules. Dissolution tests were performed using solutions of pH 1.2 and 5.6 to simulate normal gastric juice and gastric anacidity, respectively. Drugs covered with oblate were tested by the paddle method, and those covered with agar jelly were tested using the rotating basket method. Dissolution of clorazepate capsules not covered with oblate increased by approximately 10% when the pH was adjusted from 1.2 to 5.6, while those of medazepam and prazepam tablets decreased by approximately 40-60%. In contrast, the dissolution decreased significantly at both pH values for each drug covered with oblate. Dissolution further decreased when the amount of oblate was doubled. No detectable dissolution of medazepam tablets or of clorazepate capsules occurred when the drug was covered with agar jelly. Dissolution of prazepam tablets covered with agar jelly was only about 10% at the end of the test. These results indicate that dissolution is slowed and prolonged when a drug is covered with oblate or agar jelly, permitting sustained release of the drug. But, it is necessary to improve a suitable method for the dissolution.

Recovery of susceptibility to penicillin G in clinical isolates of Streptococcus pneumoniae despite increased accumulation of pbp gene alterations.

Two hundred consecutive clinical isolates of Streptococcus pneumoniae isolated in 2005 and 2006 were analysed for susceptibility to various antimicrobials, pbp gene alterations and macrolide resistance gene expression (2007 analysis) and the results were compared with previous data (2003 analysis). The average minimum inhibitory concentration (MIC) of penicillin G in isolates with 1a(m)/2x(m)/2b(m) decreased from 1.135+/-0.503 mg/L in the 2003 analysis to 0.872+/-0.540 mg/L in the 2007 analysis (P=0.0046). The prevalence of isolates with 1a(m)/2x(m)/2b(m) increased from 30.5% to 32.3%, but the difference was not statistically significant (P=0.6979). The prevalence of isolates with a clarithromycin MIC > or = 1.0mg/L increased from 65.9% to 80.0% (P=0.0005). Isolates expressing ermB increased from 46.6% to 62.6% (P=0.0004). We conclude that the decrease in penicillin resistance of S. pneumoniae does not correlate with a decrease in pbp mutations; on the contrary, the prevalence of isolates with pbp mutations increased. A decrease in penicillin resistance in S. pneumoniae with pbp mutations appears to explain the present results regarding the recovery of penicillin susceptibility. Our results suggest that the spread of mutated pbp genes among S. pneumoniae itself is not responsible for acquisition of the penicillin-resistant phenotype. Use of beta-lactams, especially oral cephalosporins, appears to be responsible for the acquisition of penicillin resistance.

Improved high-performance liquid chromatographic detection of paclitaxel in patient's plasma using solid-phase extraction, and semi-micro-bore C18 separation and UV detection.

Although a number of analytical methods for taxanes have been published, none of them are sufficiently suitable for use in a medical setting. In this study, we established an improved analytical HPLC/UV detection method using a Sep-Pak C18 cartridge for extraction and a semi-micro-borecolumn for separation. This method employed here reduced chromatographic background signals, and allowed a more sensitive analysis of taxanes in human blood sample. The recovery of taxanes after the solid-phase extraction procedure was over 90%. Chromatographic separation of paclitaxel and docetaxel was achieved within 30 min with no interference peak by a semi-micro-bore column, packed either with C18 (Wakosil 5C18 RS) or pentafluorophenyl (Curosil/Taxol) materials. The method was reproducible with coefficients of variation less than 6%. This analytical procedure was simple and sensitive with lower quantification limit of 3 ng/ml. The improved sensitivity achieved by the popular HPLC/UV apparatus, which is available in hospitals, would vouch safer and more efficient therapy with taxane.

Tandospirone, a 5-HT1A agonist, ameliorates movement disorder via non-dopaminergic systems in rats with unilateral 6-hydroxydopamine-generated lesions.

Serotonin 1A (5-HT1A) receptors are distributed throughout the brain with their highest concentrations in the frontal cortex, subthalamic nucleus and entopeduncular nucleus as well as the dorsal and median raphe nucleus. There is growing evidence that 5-HT1A receptor agonists have an antidepressant effect in individuals with major depressive disorders. Recent clinical studies suggest that tandospirone, a highly potent and selective 5-HT1A receptor agonist used clinically as an antidepressant in Japan and China, may act as an antiparkinsonian drug. In the present study, we investigated the effect of tandospirone on contralateral rotational behavior in a unilateral hemiparkinsonian rat model produced with 6-hydroxydopamine (6-OHDA). Tandospirone, as well as 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), significantly increased contralateral turnings in a dose-dependent manner (0.5-10 mg/kg). Tandospirone also remarkably potentiated the contralateral turning induced by 0.025 mg/kg of apomorphine. Pretreatment with WAY-100635, a 5-HT1A receptor antagonist, almost completely blocked the contralateral turning behavior evoked by tandospirone and 8-OHDPAT, but not that by apomorphine. SCH-23390, a selective dopamine D1 receptor antagonist, did not affect on the tandospirone-induced rotational behavior. These results suggested that tandospirone could act on postsynaptic 5-HT1A receptors and modulate excitatory amino acid pathways in the basal ganglia. Thus, tandospirone could have therapeutic potential for the treatment of Parkinson's disease by modulating neuronal activities of non-dopaminergic pathways.

Automation in drug inventory management saves personnel time and budget.

Automation in the drug distribution processes is helpful to pharmacists in creating new clinical services. We have ameliorated the drug inventory control system seamlessly connected with the physician order-entry system. This control system application, named Artima, allows inventory functions to be faster and more efficient in real time. The medicines used in our hospital are automatically fixed and arranged to sold-packages, and are ordered from each wholesaler by a fax-modem every day. Artima can search the lot number and expiration date of drug in the purchase and delivery records. These functions are powerful and useful in patient's safety and cost containment. We surveyed the inventory amount stored in the computer database, and evaluated time required for inventory management by tabulating working records of employees during past decades. Inventory decreased by 70% along with the continuous improvement of the system during the past decade. The workload in the inventory management in each section of the Pharmacy Department as well as in clinical units was dramatically reduced after the implementation of this system. The automation system in the drug inventory management allows creating new clinical positions for pharmacists. This system also could pay for itself in time.

[Sex identification from DNA in old teeth].

The sex determination was carried out on 80 fresh and 15 old teeth by amplifying sex chromosome specific sequences with the polymerase chain reaction. The DNA content in the tooth significantly decreases with aging. There was no correlation between days after evulsion and the amounts of DNA extracted from fresh teeth that had been preserved less than 186 days. The sex determination based on fresh teeth was successful using multi locus markers, DYZ-1 and DYZ-3 in combination with DXZ-1. However, amelogenin and pseudoautosomal boundary, both that are single locus markers and specific for both sex chromosomes with different lengths, could not be detected in three samples, of which DNA contents were extremely low. However, the sex determination by amelogenin amplified with fluorescent probes was possible in these three samples. We also determined sexes of 30 old specimens (15 teeth and 15 bones) from 15 human skulls using sex chromosomes locus markers. Prior to molecular sex determination, two forensic specialists determined the sex of the skull morphologically. From the 15 skulls, sex identification using multi locus marker (DYZ-1 or DYZ-3) was possible for 12 of 15 teeth and 7 of 15 bones. The sex was successfully determined from 11 teeth and 9 bones by amplification of the amelogenin locus. However, the coincidence rate of the molecular test with morphological examination was < 20%. In conclusion, sex determination on the fresh tooth would be successful using any sex chromosome marker. However, in cases on samples that have spent considerable years in the ground, pollution and putrefaction, especially, by humicolous, must be considered. Thus, sex determination by DNA testing should be regarded as accurate, when the results from two or more molecular markers are coincident. Hokkaido J Med Sci 80(2), 191-199, 2005

Genotyping of cytochrome p450 isoform genes is useful for forensic identification of cadaver.

Tailor-made medical treatment based on the polymorphism of genes encoding drug-metabolizing enzymes has been advocated and is being tried on an experimental basis at numerous centers. If DNA polymorphism analysis becomes routine in tailor-made medical treatment, it will be very useful in forensic identification. In this study, we determined the genotype frequencies of five p450 (CYP) isoform genes, CYP1A2, CYP2D6, CYP2E1, CYP2C9 and CYP2C19 in 196 Japanese individuals to evaluate their forensic usefulness. These genes encode the most important enzymes among the CYP superfamily that metabolize clinically used drug. The frequency of each allele agreed well with those reported previously and their genotype frequencies did not deviate from those expected from Hardy-Weinberg equilibrium. CYP2C subfamilies such as CYP2C9 and CYP2C19 on chromosome 10 showed high sequence homology, as high as over 95% in the regions flanking polymorphic sites. Although 3240 genotype combinations of these five CYP isoform genes are theoretically possible, 101 combinations were detected in this study. The genotype frequencies of these five isoform genes excluded their linkage. The following two genotype combinations showed the highest frequency of 0.036: CYP1A2*1A/*1A, CYP2D6*1/*10, CYP2E1*1/*1, CYP2C9*1/*1 and CYP2C19*1/*1 and CYP1A2*1A/*1C, CYP2D6*1/*10, CYP2E1*1/*1, CYP2C9*1/*1 and CYP2C19*1/*1. Thus, genotyping of CYP isoform genes should be useful in forensic identification.

Protective effects of quinaprilat and trandolaprilat, active metabolites of quinapril and trandolapril, on hemolysis induced by lysophosphatidylcholine in human erythrocytes.

We examined the effects of the angiotensin converting enzyme (ACE) inhibitors captopril, enalaprilat, quinapril, and trandolapril, and their active metabolites quinaprilat and trandolaprilat, on hemolysis induced by lysophosphatidylcholine (LPC) in human erythrocytes. LPC induced hemolysis at the concentrations above the critical micelle concentration (4 microM). Propranolol, used as a reference drug, attenuated the 50% hemolysis induced by 6 microM LPC at concentrations ranging from 100 nM to 100 microM. Similarly, quinaprilat (10 microM) and trandolaprilat (10, 100 microM) significantly attenuated the LPC-induced hemolysis, but other ACE inhibitors did not. Since propranolol possesses a membrane stabilizing action correlated with high lipophilicity, it appears that the high lipophilicity of quinaprilat or trandolaprilat is responsible for the protection from the damage induced by LPC. However, quinapril and trandolapril were not effective, although both drugs have higher lipophilicity than quinaprilat and trandolaprilat. Hence, it is suggested that the high lipophilicity alone may not contribute to the protective effects of ACE inhibitors against LPC-induced hemolysis. None of ACE inhibitors attenuated the hypotonic hemolysis (60 mM NaCl), although propranolol did. Furthermore, neither propranolol (100 microM) nor quinaprilat (50 microM) and trandolaprilat (50 microM) affected LPC micelle formation, suggesting that these drugs do not directly bind to LPC. We therefore believe that the protective effects of quinaprilat and trandolaprilat on the LPC-induced hemolysis may be related physicochemically to their highly lipophilic and ACE inhibitory structures, which probably maintain erythrocyte membrane integrity by a mechanism other than ACE inhibition, prevention of LPC micelle formation or protection against osmotic imbalance.

N-methylation underlying Parkinson's disease.

The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) leads to the hypothesis that Parkinson's disease (PD) is maybe initiated or precipitated by environmental or endogenous toxins by the mechanism similar to that of MPTP in genetically-predisposed individuals. Endogenous analogs of MPTP, such as beta-carbolines (betaCs) and tetrahydroisoquinolines, have been proposed as possible causative candidates causing PD and are bioactivated into potential neurotoxins by N-methylation enzyme(s). These N-methylated betaCs and tetrahydroisoquinoline have been higher cerebrospinal levels in parkinsonian patients than age-matched controls. Thus, there is a hypotheses to influence the pathogenesis of PD, that is, the excess enzyme activity to activate neurotoxins, such as N-methyltransferase, might be higher in PDs. Indeed, simple betaCs, via N-methylation steps, induced bradykinesia with the decreased dopamine contents in the striatum and midbrain in C57/BL mice. In younger (65 years old) PD patients, the excretion amount of N(1)-methyl-nicotinamaide was significantly higher than that in younger controls. The protein amount of nicotinamide N-methyltransferase (NNMT) was also significantly higher in younger PD patients than that in younger controls. These findings described here would indicate that the excess N-methylation ability for azaheterocyclic amines, such as betaCs, before the onset had been implicated in PD pathogenesis. On the other hand, the contribution of aberrant cytochrome P450 or aldehyde oxidase activity acting on the pyridine ring, that could act as detoxification routes of endogenous neurotoxins, would be small in the etiology of PD.